A Diecast Mineralization Process Forms the Tough Mantis Shrimp Dactyl Club

A diecast mineralization process forms the tough mantis shrimp dactyl club

Shahrouz Aminia, Maryam Tadayona, Jun Jie Lokea, Akshita Kumara, Deepankumar Kanagavela, Hortense Le Ferranda, Martial Duchampb, Manfred Raidac, Radoslaw M. Sobotad, Liyan Chend, Shawn Hoone, and Ali Misereza,f,1

aCentre for Biomimetic Sensor Science, School of Materials Science and Engineering, Nanyang Technological University (NTU), 639798 Singapore; bSchool of Materials Science and Engineering, NTU, 639798 Singapore; cLife Science Institutes, Singapore Lipidomics Incubator, National University of Singapore (NUS), 117456 Singapore; dFunctional Proteomics Laboratory, Institute for Molecular, Cell, and Development Biology, Agency for Science, Technology, and Research (A*Star), 138673 Proteos, Singapore; eMolecular Engineering Laboratory, Biomedical Sciences Institutes, A*Star, 138673 Proteos, Singapore; and fSchool of Biological Sciences, NTU, 637551 Singapore

Edited by Lia Addadi, Weizmann Institute of Science, Rehovot, Israel, and approved March 19, 2019 (received for review October 2, 2018) Biomineralization, the process by which mineralized tissues grow We used the dactyl club of stomatopods (mantis shrimps) as a and harden via biogenic mineral deposition, is a relatively lengthy model structure to study the entire formation of hard and tough process in many mineral-producing organisms, resulting in challenges apatite-based mineralized appendages. The club is a biological to study the growth and biomineralization of complex hard miner- hammer used by stomatopods to fracture the hard shells of their alized tissues. Arthropods are ideal model organisms to study preys and has emerged in recent years as a fascinating model biomineralization because they regularly molt their exoskeletons structure of bioinspired materials (6–10). The club is the most and grow new ones in a relatively fast timescale, providing oppor- mineralized appendage of the dactyl segment and exhibits a tunities to track mineralization of entire tissues. Here, we monitored complex architecture across multiple length scales, allowing the the biomineralization of the mantis shrimp dactyl club—amodel animal to deliver extremely high impact forces against its targets bioapatite-based mineralized structure with exceptional mechanical without sustaining macroscopic fracture. In brief, the dactyl club — properties immediately after ecdysis until the formation of the fully is a multilayer composite at the mesoscale that can be broadly functional club and unveil an unusual development mechanism. A separated into an outer region that expands toward the impact flexible membrane initially folded within the club cavity expands to surface and an inner bulk region. Both regions exhibit distinct BIOPHYSICS AND form the new club’s envelope. Mineralization proceeds inwards by chemical compositions and microstructures. The outer region is COMPUTATIONAL BIOLOGY mineral deposition from this membrane, which contains proteins reg- mostly made of crystalline fluorapatite (FAP) nanorods that are ulating mineralization. Building a transcriptome of the club tissue and preferentially oriented perpendicular to the impact surface, with probing it with proteomic data, we identified and sequenced Club a small presence of calcium sulfate (7). Moving toward the bulk, Mineralization Protein 1 (CMP-1), an abundant mildly phosphorylated crystallinity of FAP decreases and the mineral phase gradually protein from the flexible membrane suggested to be involved in calcium phosphate mineralization of the club, as indicated by in vitro transitions toward amorphous calcium phosphate (ACP). The inner bulk region contains both ACP as well as amorphous cal-

studies using recombinant CMP-1. This work provides a comprehen- BIOCHEMISTRY sive picture of the development of a complex hard tissue, from the cium carbonate (ACC) that decorate chitin fibrils arranged in a secretion of its organic macromolecular template to the formation of the fully functional club. Significance

biomineralization | bioapatite | ecdysis | stomatopod dactyl club | Monitoring hard tissues calcification using vertebrates is chal- mineralization proteins lenging, owing to the internal location and slow biomineralization process of these tissues. Crustaceans are ideal model ard mineralized tissues grow through biogenic mineral de- organisms to overcome this challenge because they regularly Hposition (biomineralization) and this process is a central molt their exoskeletons. Using the ultratough mantis shrimp attribute of vertebrate development (1). However, investigating dactyl club as a model biomineral, we detect all stages during the growth process of entire hard tissues in vertebrates such as the development of a calcified tissue, from secretion of the bone or teeth is challenging, owing to the relatively long timescale organic template that regulates mineral deposition to matu- ration of the functional club. We unveil a peculiar growth over which mineralized tissues are formed (2, 3) and to sample mechanism: a flexible membrane initially folded in the club availability. In contrast, crustaceans are convenient model organ- cavity expands after ecdysis to form the new club outer en- isms to study biomineralization because they regularly shed their velope from which biomineralization proceeds. A main phos- mineralized exoskeletons (cuticles) and grow new ones through phorylated protein within that membrane is sequenced and molting cycles (4, 5). Specifically, molting and calcification of cu- demonstrated to regulate mineral crystal growth. ticles occur in just a few days or weeks, providing the distinctive opportunity to follow the entire biomineralization process for Author contributions: S.A. and A.M. designed research; S.A., M.T., J.J.L., A.K., D.K., H.L.F., model organisms that can be maintained in the laboratory. M.R., R.M.S., L.C., and S.H. performed research; S.A., J.J.L., A.K., H.L.F., M.D., M.R., R.M.S., Molting, the shedding (or ecdysis) of the exoskeleton, is an L.C., S.H., and A.M. analyzed data; and S.A. and A.M. wrote the paper. essential event of arthropod development, during which the hard The authors declare no conflict of interest. exoskeleton is replaced with a fresher, slightly larger one to ac- This article is a PNAS Direct Submission. commodate the animal’s growth. Following molting, the freshly Published under the PNAS license. formed exoskeleton is still soft and cannot fulfill its function, Data deposition: Transriptomic data of O. scyllarus dactyl club have been deposited in the NCBI BioProject (accession no. PRJNA528158. Proteomic data have been deposited in namely providing a protecting barrier against predators, pathogens, the jPOST Repository, https://repository.jpostdb.org (accession no. JPST000563), and in the or the natural environment. Whereas molting takes just a few mi- ProteomeXchange Consortium database (accession no. PXD013153). nutes, mineralization of the new exoskeleton is longer, from days to 1To whom correspondence should be addressed. Email: [email protected]. weeks. Nevertheless, compared with vertebrate mineralization, the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. process is short enough such that the different stages can be studied 1073/pnas.1816835116/-/DCSupplemental. in the laboratory with convenient model organisms (5). Published online April 11, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1816835116 PNAS | April 30, 2019 | vol. 116 | no. 18 | 8685–8692 Downloaded by guest on September 29, 2021 helicoidal pattern (6, 10). Collectively this multilayer design en- club, mostly composed of organic phases, as shown by the high dows the club with exceptional tolerance against contact stresses carbon content and the absence of calcium. We also note the pres- (8) and serves as inspiration for the design of damage-tolerant ence of sulfur in the premolt membrane, which may act as a reservoir biocomposites (11, 12). for calcium sulfate that is also found in the fully formed clubs (7). The fresh cuticle does not provide protection against external Molting Stages of the Mantis Shrimp threats: although the expanded membranes displayed the overall Mantis shrimps shed their exoskeletons a few times per year. During geometry of a mature club, they were not functional due to their this process, they are vulnerable to attacks from other predators weak mechanical properties (they could easily be bent and torn by (such as crabs, their congeners, or starfish) since their raptorial hand), which explains why mantis shrimps refuse to hit any target appendages are not functional for either hunting or defense pur- and hid inside their nest after ecdysis. Since their survival depends poses. To mitigate this drawback, in the premolt stage, mantis on a fully functional dactyl club, they must rapidly build a new one, shrimps secure a nest by shattering rocks, shells, and corals and then thus providing a unique opportunity to study the entire bio- collect the broken pieces to build a protecting nesting cavity (SI mineralization process. In our aquaria containing artificial seawater, Appendix,Fig.S1A–C). During this process, the dactyl clubs are we found that a partially functional club was formed within a week. eroded on their impact surface due to high-energy hits against rock- solid targets (SI Appendix,Fig.S1E and F), though they do no Formation of the Club by a Diecast Mechanism sustain catastrophic fracture. Subsequently during the molting pe- We followed our initial observations of the molting process with riod, stomatopods hide in this nest and avoid external contact. systematic investigations of the different stages of dactyl club Our initial observations (SI Appendix, Movie S1) revealed that formation right after ecdysis by examining the club structure and during molting, the entire cuticle including the eye cups and the mechanical properties at different development stages, which dactyl clubs were shed and buried in the sand for partial reuti- revealed an unusual formation mechanism that, to the best of lization of nutrients (5). Fig. 1A is a picture of a mantis shrimp our knowledge, has not been previously reported in highly min- immediately after molting next to the old cuticle. We found that eralized hard tissues. In clear contrast with the hypothesis of an while the new exoskeletons were slightly larger than those before inside-to-outward growth (13), we found that the club is built by molting, the new dactyl clubs initially appeared as folded mem- an outside-to-inward mechanism as corroborated by optical and branes that were much smaller than the shed dactyl clubs (exuviae, scanning electron microscopy (SEM) images of dactyl clubs at Fig. 1 B and C). However, these folded membranes expanded various development stages shown in Fig. 2. Within the first hour during the first hour after ecdysis to form the outer shape of the after ecdysis, the folded membrane expanded to form the outer new dactyl clubs. Once expanded these membranes were larger geometrical shape of the new club (Fig. 1D). This expanded than the exuviae but soft and flexible (Fig. 1D) and were filled membrane was initially flexible but became a delicate and with hemolymph fluid (“crustacean blood”). brittle shell on the second day. We also observed the presence A closer look revealed that these folded membranes were of microchannels on day 2 (Fig. 2B, Middle Left and SI Ap- initially stored inside the internal cavity of the old clubs (Fig. 1 B pendix,Fig.S2B) that likely enables the continuous growth of and C) before their rapid expansion, which occurred within the the outer layer by providing diffusion paths for the delivery of first few minutes following ecdysis. These observations indicated ionic species and/or nanocluster precursors (14) required for that these membranes act as templates for club formation, and mineral deposition. that they must be folded and flexible to allow easy extrusion The formation of the inner, less mineralized layer of the club from the internal cavity of the old club. Energy dispersive X-ray started after a few days and included the deposition of nano- spectroscopy (EDS) elemental mapping of a dactyl club cross- fibrils (Fig. 2 A and B, Middle Right) that form the helicoidal sectional cut (SI Appendix, Fig. S2A) confirmed the presence of chitinous-based microstructure previously reported (6). After the premolt membrane inside the internal cavity of the old dactyl 1 wk, the dactyl club became functional and the animals started

Fig. 1. Ecdysis process of the mantis shrimp. (A) Ecdysis of the entire mantis shrimp cuticle (Left), including the dactyl clubs (Right). The exuvia (shed cuticle) is seen next to the animal. (B) Exuvia and new appendages dissected from a mantis shrimp during ecdysis showing the folded membrane that was stored inside the old dactyl club. (C) Micro-CT image of an old dactyl club illustrating the presence of the internal cavity inside the dactyl club. (D) Within an hour, the folded membrane (Left) expands to take the shape of the new dactyl club (Middle), which is still soft and flexible (enlarged image Right) and contains the hemolymph fluid made mostly of hemocyanin (SI Appendix, Fig. S3 for proteomic analysis).

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Fig. 2. Structure of the dactyl club at various time points of the molting and formation process (premolt, and 2 d, 1–2 wk, and 1 mo after molting). (A) Optical micrographs of cross-sectional cuts of entire clubs. (B) Low-magnification (Upper) and higher-magnification (Lower) SEM micrographs. The club grows by an outside-to-inward mechanism the first month following ecdysis. It starts with expansion of the premolt membrane within the first hours following ecdysis to form the overall envelope of the new dactyl club. (C) Carbon elemental map of a mature club before molting, showing the presence of a premolt

membrane inside the internal club cavity. Adapted from ref. 7. (D) Schematic of the formation process of the dactyl club during the first month after ecdysis. BIOCHEMISTRY

venturing out of their nest to hunt for food and reengage in colocalized with FAP crystallites (7), which was detected 2 d after striking actions. Finally, after about 1 mo the dactyl clubs were ecdysis (Fig. 3A, Inset). EDS analysis confirmed the absence of Ca fully formed and exhibited the complex multiscale architecture and P in the premolt membrane and immediately after expansion with their characteristic high impact tolerance (6, 7) (Fig. 2 A and (Fig. 3B), but both elements increased after 24 h with a Ca/P weight B, Right). These observations can be summarized in the schematic ratio of ∼1.7, corresponding to calcium phosphate. In parallel we cartoon shown in Fig. 2D. The folded membrane rapidly expands evaluated the mechanical properties of the club at various de- after ecdysis, an unfolding process that determines the final outer velopmental stages by nanoindentation and found that the ex- shape of the dactyl club. Club formation is then initiated from this panded membrane in hydrated conditions was soft and flexible outer envelope and proceeds toward the inner layer, a mechanism (elastic modulus E = 0.12 ± 0.03 GPa). After 48 h, the modulus resembling diecast processing. In the final stage, a new internal strongly increased by nearly two orders of magnitude (E = 10.2 ± cavity of the club is maintained, which will serve as a template for 2.2GPa)andafter1wkE reached 53.5 ± 3.6 GPa, which is ∼85% the formation of the next membrane and club. that of mature dactyl clubs (E = 62.8 ± 1.9 GPa). Structural Formation and Mechanical Stiffening In Vitro Apatite Biomineralization Concomitant to microscopic observations, we also probed the Since biomineralization of the club was clearly initiated from the structural and mechanical properties of the club at various de- expanded membrane, we hypothesized that the membrane con- velopment stages (from a few hours until 1 wk) using Raman tains the proteins that template and regulate apatite nucleation spectroscopy, EDS analysis, and nanomechanical measurements, and growth during mineralization of the club. To test this hy- and the data were compared with those from fully formed clubs. pothesis, we conducted an in vitro mineralization assay designed In the expanded membrane, only peaks associated with organic after ref. 15 and incubated a membrane in a buffer saturated ∼ −1 −1 2+ 3− phases were detected at 1,160 cm and 1,520 cm (Fig. 3A), with Ca and PO4 ions for 7 d (Fig. 4A), after which the in- which can be assigned to proteins and chitin. On the other hand, cubated membrane was thoroughly rinsed and probed by SEM, no peaks related to calcium phosphate minerals were observed. EDS, Raman spectroscopy, and nanoindentation. However, 2 d after ecdysis the characteristic ν1 peak of apatite at We first verified the absence of calcium phosphate in the fresh − 965 cm 1 was detected with weak intensity, suggesting that min- and rinsed expanded membrane by Raman spectroscopy (Fig. eral deposition had been initiated. The ν1 peak intensity strongly 4C), which was further validated by EDS measurements that increased after 1 wk and was the most prominent, with the overall confirmed the absence of Ca (Fig. 4D). After incubation, Raman spectrum similar to that of the fully formed clubs in the outer re- spectra clearly indicated the presence of crystalline calcium phos- 3− gion near the impact surface (Fig. 3A). We also noted the presence phate with the appearance of the intense PO4 ν1 vibrational peak −1 −1 of calcium sulfate (ν1 peak at 1,007 cm ) previously found to be at 965 cm . SEM observations showed the presence of spherical

Amini et al. PNAS | April 30, 2019 | vol. 116 | no. 18 | 8687 Downloaded by guest on September 29, 2021 as P and Ca storage to accelerate club mineralization after molting. 2+ 3− After incubating the hemolymph with Ca /PO4 saturated solution and washing the samples, a high content of Ca and P remained within these granules as evidenced by the strong signal intensity of Ca and P EDS peaks under identical acquisition conditions (SI Appendix,Fig.S6), possibly pointing out toward a storage role of mineralization ions for the hemolymph. Additionally, the granules were amorphous, as no evidence of crystallinity was detected by electron diffraction (SI Appendix,Fig.S6C). Identification and Sequencing of Proteins Controlling Apatite Nucleation and Growth Having established that proteins within the flexible membrane could regulate apatite nucleation and growth, we sought to iden- tify and sequence these putative mineralization proteins using a combined transcriptomic/proteomics strategy we previously de- veloped (20–22). First, transcriptome libraries (23) were assem- bled from mRNAs extracted from the epithelial cells of mature clubs of adult mantis shrimps as well as from larvae that were reared in our laboratory. Since there are no reference genomes for stomatopods, we generated transcript databases by de novo transcript assembly with the Trinity software suite (24). In parallel, we extracted proteins from flexible membranes immediately after ecdysis using a guanidine thiocyanate-based buffer to maximize protein extraction. SDS polyacrylamide gel electrophoresis (SDS/ PAGE) of the extracts revealed broad faint bands in the 15- to 20- kDa range as well as a sharp faint band between 60 and 75 kDa (Fig. 5A). These bands were excised from the gel and digested with trypsin. Subsequently, tryptic peptides were extracted from the gel slices and separated on a nano C18 high-performance liquid chromatography (LC) column and subjected to LC tan- Fig. 3. Structural and mechanical properties of the dactyl club at different dem mass spectrometry. De novo sequencing of the tryptic pep- development stages. (A) Raman spectra at different stages of formation. FAP tides was obtained using the PEAKS studio 8.0 software (25). De −1 (ν1 vibration mode at 965 cm ) is not detected in the expanded membrane, novo peptide fragments identified with PEAKS were then screened weakly observed in the 24- to 48-h postmolt club, and abundant in the 7-d directly against the transcriptome libraries of the club using the postmolt club. (B) EDS point analysis at different club formation stages Spider and PEAKS search routines. (variation in Ca from 24-h postmolt onward is attributed to statistical vari- We further narrowed down the transcriptome screening of ability between different animals). (C) Elastic modulus (E) of the club (outer proteins detected by de novo sequencing with a combination of region) at different stages of formation. The expanded membrane in the the following criteria: (i) molecular weight (MW) larger than hydrated state is soft (E = 0.12 ± 0.03 GPa). Two days after ecdysis (newly formed club), E increases to ∼10.2 ± 2.2 GPa. After 2 wk (immature club) E = 50 kDa to match the main sharp band detected by SDS/PAGE; 53.5 ± 3.6 GPa, close to E in mature clubs (E = 62.8 ± 1.9 GPa). (ii) high transcript levels; (iii) enriched with acidic residues since calcium phosphate mineralization is well established to be con- trolled by highly acidic proteins (26, 27); and (iv) presence of chitin- particles at ∼100 nm on the fractured surface of the membrane binding domains, since the flexible membrane is a protein/chitin (Fig. 4B). Point analysis of these particles by EDS (Fig. 4D) complex. We identified five proteins containing chitin-binding revealed a high intensity of Ca and P elements that corroborated domains in the club transcriptome (SI Appendix,TableS1and a the formation of calcium phosphate. Furthermore, the elastic full list of tryptic peptides shown in SI Appendix,TableS2), one of modulus (hydrated conditions) of the membrane increased up to which was abundant with acidic residues [8.3 mol% aspartic acid 20-fold after in vitro mineralization (Fig. 4E). As a control, the (Asp) and 4.1 mol% of glutamic acid (Glu)], with no homology to identical incubation experiment was conducted on a membrane any known protein, which we termed Club Mineralization Protein that was previously subjected to an alkali peroxidation treatment 1 (CMP-1). We obtained the full-length sequence of CMP-1 by to remove proteins, so that only chitin was left as the organic RACE-PCR (28) using the cDNA library of the club as a template for PCR, and the final gene was sequenced with Sanger se- phase in the membrane (9). No calcium phosphate formation was quencing. Both the signal peptide as well as a stop codon in the observed after 7 d of incubation as evidenced by EDS measure- gene encoding CMP-1 were detected, confirming that the full- SI Appendix ments ( ,Fig.S4). length sequence of CMP-1 was achieved. The MW of CMP-1 is In addition, we also found that the flexible membrane imme- 65 kDa, matching the MW of the sharp band detected by SDS/ diately after molting was filled with the hemolymph fluid (Fig. 1D), PAGE from the flexible membrane extract (Fig. 5A). which mostly comprises hemocyanin as confirmed by a compre- To confirm that this band corresponded to CMP-1, we con- hensive set of proteomic analyses of the fluid (SI Appendix,Fig.S3). ducted additional protein extraction from a flexible membrane Previous studies have suggested that crustacean hemocyanin is not using a sonication method and ran the extract by SDS/PAGE (SI restricted to its well-known oxygen-carrying role but may also be Appendix,Fig.S7A), followed by LC MS/MS analysis of individual involved in calcium storage (16, 17) and transport to the growing excised bands (29). CMP-1 tryptic peptides were identified (SI cuticle (18). For example, we observed by transmission electron Appendix, Table S3) with a protein coverage of 39% (SI Appendix, microscopy (TEM) dense amorphous granules ∼50–100 nm in Fig. S7C), therefore corroborating the assignment of CMP-1 to diameter in the native hemolymph, which were enriched with Ca the sharp band between 50 and 75 kDa. All detected peptides and P (SI Appendix,Fig.S5). These granules may be condensed were from the N terminus due to the absence of cleavage sites in + phosphates forming complexes with Ca2 ions (19) that may serve the C terminus. Furthermore, we found by using quercetin-based

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Fig. 4. In vitro mineralization studies using the native proteins of the dactyl club as template for biomineralization. (A) Expanded membrane forming the 2+ 3− outer envelope of the club by incubation of the flexible extended membrane in buffer saturated with Ca and PO4 ions. (B) Pre- and postincubation SEM 2+ 3− images revealing the flexible and fibrous structure of the expanded membrane, which turned into a rigid structure after incubation in Ca /PO4 saturated 2+ 3− solution. (C and D) Raman (C) and EDS (D) spectra of an expanded membrane and a membrane incubated in Ca /PO4 saturated solution. No trace of 3− mineralization in the extended membrane was detected (no PO4 band in the Raman spectrum, and no Ca peak in the EDS spectrum). The incubated 3− membrane, on the other hand, was mineralized as indicated by the presence of PO4 bands (ν1, ν2, and ν4 in the Raman spectrum) and by the strong intensity of Ca and P peaks in the EDS spectrum. (E) The elastic modulus (E) of the membrane (hydrated conditions) increased up to 20-fold after 1 wk of incubation in 2+ 3− the Ca /PO4 saturated solution from E = 0.12 ± 0.03 GPa to E = 2.16 ± 0.3 GPa, and from E = 0.16 ± 0.03 GPa to E = 1.44 ± 0.3 GPa, for samples 1 and 2, respectively.

phosphostain (SI Appendix, Fig. S7B) that CMP-1 was mildly 17 amino acid long and is comprised of 14 Asp intervened with phosphorylated, and two phosphorylated Ser residues (S55 and three Phe residues. Similar to osteopontin and other proteins in- S226) were detected by LC MS/MS with high confidence (SI volved in biomineralization (31), it is noteworthy that CMP-1 is Appendix, Fig. S7 C and D). CMP-1 harbors several sequence also phosphorylated, although to a much lesser extent with only characteristics that make it particularly suitable as an organic two detected sites at S55 and S226. Additional phosphorylated template for biocomposite mineralization. First, it features a central sites may also be present in the C terminus; however the lack of domain (R294–Y339) with sequence homology to the chitin-binding cleavable tryptic peptides in this region precluded us from 4 superfamily (Fig. 5B), which was predicted to bind to chitin, based identifying them. on molecular dynamic (MD) simulations (Materials and Methods and SI Appendix,Fig.S8). Second, it contains a very acidic N- In Vitro Apatite Mineralization and Microdroplet Formation + terminal domain that we hypothesized could bind Ca2 ions to from Recombinant CMP-1 control calcium phosphate nucleation. This dual binding role to both To confirm our hypothesis that CMP-1 can nucleate and regulate chitin nanofibrils (to form the organic template of the flexible mem- apatite formation, we cloned, expressed, and purified CMP-1 to + brane) and to Ca2 for subsequent mineral deposition is reminiscent homogeneity (Fig. 5C) and then conducted in vitro assays by + of GAP65 identified in the gastrolith of the crayfish that stabilizes incubating soluble recombinant CMP-1 (rCMP-1) in a Ca2 / 3− amorphous calcium carbonate (30). Notably, the N-terminal do- PO4 saturated solution using similar protocols as for the native main contains two poly-Asp rich motifs that are strikingly similar to membrane. After 6 d of incubation, we observed rod-like min- poly-Asp domains found in osteopontin, a main protein regulating erals by SEM (Fig. 5D), which were composed mainly of Ca and apatite mineralization in bone (31, 32). The first motif (D154–D175) P as inferred from EDS, with a Ca/P ratio of 1.72, close to the is 22 amino acid long and contains 17 Asp residues with the longest value (1.68) documented for crystalline apatite. To gain further stretch comprising 10 Asp. The second motif (D229–D246)is details on the nucleation and growth of these rods, we conducted

Amini et al. PNAS | April 30, 2019 | vol. 116 | no. 18 | 8689 Downloaded by guest on September 29, 2021 Fig. 5. CMP-1 sequence and in vitro mineralization. (A) SDS/PAGE of protein extract from the flexible membrane. The arrow indicates CMP-1 as confirmed by MS/MS analysis of individual bands conducted on additional gel extracts (SI Appendix, Fig. S7A). This band also stained positively for the quercetin phosphostain (SI Appendix, Fig. S7B) indicating that CMP-1 is mildly phosphorylated. (B) Full-length sequence of CMP-1, obtained by searching flexible membrane peptides identified by MS/MS against the club tissue transcriptome, followed by RACE-PCR. *Phosphorylation sites detected by MS/MS. Bioinformaticpredictions are underlined in yellow (β-sheets) and blue (α-helices), or highlighted in green (chitin-binding domain). Asp-rich cluster regions that likely bind Ca2+ ions are highlighted in red. The structure of the chitin-binding domain computed by MD simulations is also shown below (see SI Appendix, Fig. S8 for more details). (C)SDS/ 2+ 3− PAGE of rCMP-1 after purification. (D) SEM image of rCMP-1 after 6 d of incubation in the Ca and PO4 saturated solution (Top)andEDSspectrum(Bottom) showing the presence of rod-like structures containing P and Ca (Na, Mg, Al, and Si are from the soda-lime glass substrate). (E)TEMimaging(Top)andelectron 2+ 3− diffraction patterns (Bottom)ofrCMP-1 incubated in the Ca and PO4 saturated solution at various time points. Amorphous nanoclusters containing Ca and P were observed at 0.5 d and 1 d. From 1.25 d and beyond crystalline apatite was observed, with increasing crystallinity over time. Full assignment of diffractions peaks is shown in SI Appendix,Fig.S9.

TEM measurements at various time points (Fig. 5E). At 0.5 d detected in our experiments before they crystallized into ori- and 1 d, we observed nanoclusters 40–60 nm in size, and these ented apatite nanorods, this suggests that CMP-1 can both nu- clusters were amorphous, based on their electron diffraction cleate transient ACP and then promote apatite crystallization patterns (Fig. 5E, Bottom). After 1.5 d, larger and elongated rods regardless of phosphorylation. Furthermore, crystalline apatite were observed, which were oriented crystalline apatite as evi- was detected early during club formation (Fig. 3A) in the outer denced by electron diffraction. After 6 d of incubation, the layer of the club (Fig. 2D), whereas ACP is not found in this layer crystallinity of the apatite rods further increased as revealed by of the club (7). Similarly in our in vitro mineralization experi- single diffraction spots on the electron diffraction pattern (for ments using the extracted flexible membrane as a template, full assignment of diffraction peaks, see SI Appendix, Fig. S9). crystalline apatite was formed after a few days of incubation but These data corroborate previous studies by Dey et al. (14) in not ACP (Fig. 4C). Since CMP-1 is abundant in the flexible which the amorphous-to-crystalline transition of calcium phos- membrane, the colocalization of crystalline apatite and CMP- phate was shown to be initiated through prenucleation of 1 suggests that the role of phosphorylation in CMP-1 may be nanoclusters. Among the invertebrates, we are only aware of one to regulate the kinetics of apatite crystallization, but this hy- other sequenced protein (from the crayfish molar tooth) that pothesis remains to be validated. To answer this question, it will also regulates apatite formation (33). We emphasize that these be critical to determine the currently unknown in vivo conditions in vitro experiments were conducted using rCMP-1, which lacks (such as pH and ionic strength) under which the club is formed phosphorylation detected in the wild-type CMP-1. Phosphory- because they likely influence protein activity (37). lated proteins in biomineralization have been shown to stabilize To assess the expression level of CMP-1, we also conducted ACP and inhibit crystallization in some cases (34, 35) or promote qPCR experiments using mRNA extracted at different stages of apatite formation in others (36). Since ACP nanoclusters were molting. We found that the expression level increased from a few

8690 | www.pnas.org/cgi/doi/10.1073/pnas.1816835116 Amini et al. Downloaded by guest on September 29, 2021 hours after molting to reach its highest level at day 2, and then study reveals a peculiar development mechanism of the club. The dropped at day 7 (SI Appendix, Fig. S10). Since the outer (im- inner cavity of the fully formed club contains a folded flexible pact) layer of the club made of crystalline apatite is fully formed membrane that rapidly expands immediately after ecdysis to within the first few days after molting, and since the remaining form the outer envelope of the club. Club mineralization ensues formation of the club mostly involves deposition of amorphous via an outside-to-inward mineral deposition mechanism, forming calcium carbonate within the interior of the club cavity (but no the functional, impact-resistant club within a few weeks. The flexible additional crystalline apatite from day 7 onwards, see Fig. 2 A outer membrane is a chitin/protein macromolecular complex whose and D), these results further support the notion that CMP-1 proteins can trigger calcium phosphate nucleation and mineral- plays an important role in regulating apatite crystallization. ization. Proteins within the flexible membrane were extracted This is also in agreement with the fact that transcript levels from the membrane and sequenced via a dual transcriptomic/ (normalized for sequencing depth) were approximately one or- proteomic approach. One of the most abundant proteins (CMP-1) der of magnitude higher in the mature club compared with the was in particular identified and its full-length amino acid sequence saddle segment of the dactyl appendage (SI Appendix, Table S1), obtained. CMP-1 comprises two key domains conferring the critical which is made of amorphous calcium phosphate but does not dual function of binding to chitin to form the organic matrix, as well + contain crystalline apatite (9), suggesting a direct correlation as to Ca2 ions to regulate calcium phosphate mineralization. In between calcium phosphate crystallinity and CMP-1 expression addition, CMP-1 is slightly phosphorylated and can phase separate level. Furthermore, transcript levels in whole larvae of mantis into liquid droplets, which are characteristics that may also play a shrimps—which do not develop a club yet—were even lower, role in the growth kinetics and polymorphism control of calcium namely 500-fold less than in the club tissue. phosphate during club formation. In vitro assays in the presence In addition to regulating apatite nucleation and growth, we of rCMP-1 resulted in the formation of apatite nanorods, sug- also observed that rCMP-1 formed liquid droplets at pH 8.2 in gesting the ability of CMP-1 to regulate crystalline apatite. Re- the presence of 5 mM CaCl2 and above, with droplet size in the vealing the natural fabrication process by which a remarkable range of 1–3 μm as revealed by SEM imaging and dynamic light tough biomineralized appendage is produced offers bioinspired scattering measurements (SI Appendix,Fig.S11). This observation lessons that may be applied in additive manufacturing of bioceramics, parallels recent findings by Bahn et al. (38) for Pfam-80 protein with potential applications for the next generation of orthopedic that controls calcium carbonate formation in oyster nacre, and or dental implants. further supports the growing concept that biomacromolecules con-

trolling biomineralization phase separate into coacervate droplets Materials and Methods BIOPHYSICS AND

that can sequestrate and concentrate inorganic ions for subsequent Detailed experimental procedures are provided in SI Appendix, Materials and COMPUTATIONAL BIOLOGY mineral deposition (38, 39). There has been mounting evidence in Methods. In brief, mantis shrimps (Odontodactylus scyllarus) were purchased recent years that proteins undergoing simple component liquid– from aquarium suppliers in Singapore, maintained in artificial seawater liquid phase separation (coacervation) are intrinsically disordered aquaria, and carefully monitored to detect molting events. Dactyl clubs were (40) or contain intrinsically disordered regions (IDRs) (41). collected at various time points during molting and formation cycles, from Inspecting CMP-1’s primary structure, we note that the C-terminal immediately (flexible membrane) up to 1 mo (mature club) after molting region is highly enriched in glycine (Gly) and alanine (Ala) amino and used for subsequent studies. Biomineral characterization was carried acids and also contains a significant amount of proline (Pro), which out by Raman confocal microspectroscopy, EDS, and optical microscopy. BIOCHEMISTRY Mechanical characterization was carried out on hydrated samples using are characteristic features of IDRs as also confirmed by our bio- depth-sensing nanoindentation. For in vitro biomineralization using native informatic predictions of this region (Fig. 5B). This suggests a role 2+ 3− flexible membranes, the latter were incubated in a Ca and PO4 saturated fortheCterminusofCMP-1toinduce coacervation as a way to buffer and analyzed postmineralization by Raman spectroscopy, EDS, and concentrate inorganic ions before mineralization. nanoindentation. Transcriptome libraries of dactyl appendage segments It is very likely that other proteins detected in our combined [namely the club (6–8) and the saddle (9)] as well as of whole larvae reared in transcriptomic/proteomic dataset are also involved in club miner- our artificial seawater aquaria were prepared from mRNA extracted from alization since it is now well established that tissue biomineraliza- tissues that were initially stored in RNAlater solution. The libraries were se- tion and crystal polymorphism is carefully orchestrated by multiple quenced on a HiSEq. 2000 Illumina sequencer and the de novo transcript as- biomacromolecules, including low MW compounds (42). For sembly was performed with Trinity (24). For CMP-1 identification and sequencing, example, we found chitin-binding proteins with homology to proteins were extracted from the flexible membrane and analyzed by SDS/ PAGE, followed by LC MS/MS of both entire gels and excised individual cuticular proteins from other crustaceans, such as the American bands. In both cases, trypsin in gel digestion was carried out, tryptic peptides lobster or the brown crab (SI Appendix, Table S1), suggesting were analyzed with either PEAKS (25) or Proteome Discover 2.2 (Thermo), that the flexible membrane is a macromolecular complex com- and then searched against our de novo transcriptome libraries. Full-length prising chitin and multiple chitin-binding proteins. Likewise, protein sequencing of CMP-1 was achieved using both 3′ and 5′ RACE-PCR there are several other peptides uncovered by de novo MS/MS with RNA extracted from the club tissue as the template. Gene encoding peptide sequencing that are enriched with acidic residues (SI CMP-1 was cloned into pET28A plasmid, transformed to Escherichia coli Appendix, Table S2) and that may also play a role in binding Ca BL21, and rCMP-1 was expressed in LB medium using isopropyl β-D-1-thio- ions and regulating apatite mineralization. Finally, phosphorylated galactopyranoside to induce expression. rCMP-1 was purified by immobilized proteins with lower MWs than CMP-1 were also detected in the metal affinity chromatography followed by FPLC and desalted before usage. In vitro mineralization assays were conducted by incubating rCMP-1 with a flexible membrane (SI Appendix,Fig.S7B), which is another 2+ 3− Ca and PO4 saturated buffer and analyzed by SEM, EDS, and TEM in both characteristic feature of proteins controlling biomineral formation the imaging and electron diffraction modes. Liquid–liquid phase separation (31). However, CMP-1 was the only protein we detected that (microdroplet formation) of rCMP-1 was achieved by pipetting the purified

harbored several clusters of acidic residues (poly-Asp) along its protein in a CaCl2-containing Tris buffer. Secondary structure predictions and primary sequence, which is a common feature associated with sequence alignment of CMP-1 were conducted using a range of bioinformatic proteins controlling apatite formation. A comprehensive proteo- tools, including PSIPRED (43), BLASTp (44), CLUSTAL W (45), and Modeler (46). mic analysis of all proteins detected in the flexible membrane is MD simulations of CMP-1 were carried out using Amber 12 (47). Chitin-CMP- currently underway and will be reported elsewhere. 1 interactions were modeled using a 3D structure of chitin from ChemSpider and Schrodinger 9.0 with previously used protocols (48). Conclusion ’ ACKNOWLEDGMENTS. We thank Isaiah Chua for helping with flexible Exploiting the fast molting process of crustaceans exoskeletons, membrane sample collection; Kong Kiat Whye for technical support with we have unveiled the entire formation of the mantis shrimp transcriptome library preparation and qPCR measurements; and the central dactyl club from ecdysis to the mature, impact-resistant club. Our Facilities for Analysis, Characterization, Testing, and Simulation at NTU for

Amini et al. PNAS | April 30, 2019 | vol. 116 | no. 18 | 8691 Downloaded by guest on September 29, 2021 access to their electron microscopy facilities. This work was funded by Sustainable Materials (IBSM, NTU). H.L.F. gratefully acknowledges the Swiss the Singapore National Research Foundation (NRF) through an individual National Science Foundation for an individual postdoctoral scholarship NRF Fellowship (to A.M.), and by the Strategic Initiative on Biomimetic and (Grant_P2EZP2_172169). R.M.S. and L.C. were supported by A*Star core funding.

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